酶促化学偶联合成多羟基聚氨酯材料及介导颗粒酶B递送的研究

开发高效、安全的递送载体，是提升颗粒酶B传输稳定性及跨膜转运能力的重要策略，是推进颗粒酶B作为癌症治疗药物的关键。本项目拟通过分子克隆技术从嗜热微生物中开发高催化活性及稳定性的嗜热酯酶，在理解酶促聚合机制的基础上，通过蛋白质工程技术进一步提升催化聚合反应的活性；以己内酯、癸二酸二乙酯、N-甲基二乙醇胺为单体，通过酶促开环聚合与缩聚反应相偶联的模式制备聚氨酯载体材料，利用原子转移自由基聚合反应对聚氨酯材料进一步修饰改性，构建多羟基聚氨酯材料；以该多羟基聚氨酯材料为载体，进行颗粒酶B分子的递送，细胞与动物水平上评价递送体系的跨膜转运能力、肿瘤增殖的抑制能力、生物分布及作用机制。本研究的实施，不仅有助于我们深入认识酶促聚合反应的机制及催化过程中酶结构与功能关系，同时提供了一条新型的绿色生物医用高分子材料制备途径，对酶促聚合技术的产业化进程及基于蛋白质递送的恶性肿瘤治疗均具有重要的推动作用。

The development of efficient and safe carriers is an important strategy to improve the stability and transmembrane ability of Granzyme B, which has been considered to be a key factor for achieving Granzyme B-based anti-tumor therapy. In this project, thermophilic esterases with favorable catalytic activity and stability are explored through molecular cloning, and then their polymerization activity is further improved through protein engineering techniques on the basis of the mechanism of enzymatic polymerization. Then enzymatic ring-opening polymerization and polycondensation are combined to construct cationic poly(amino-co-ester) using caprolactone, diethyl sebacate and N-methyldiethanolamine as monomers. Further, multihydroxyl poly(amino-co-ester) is synthesized through the modification of polymer by atomic transfer radical polymerization. The obtained poly(amino-co-ester) derivative is used as the carrier in the Granzyme B delivery to evaluate the transmembrane ability, inhibition of tumor proliferation, biodistribution and mechanism of nanocomplex at the cellular and animal levels. The project will be useful to get a deeper insight into the mechanism of enzymatic polymerization and the structure-function relationship during enzymatic catalysis, and also provides a green route for the preparation of biomedical polymers. Thus, it is of great significance for facilitating the enzymatic polymerization at an industrial scale and the tumor therapy based on protein delivery.